Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system (CNS); Forsythe and Westbrook, J. Physiol. (Lond). 396:515-533 (1988) and Hestrin, et al., J. Physiol. (Lond). 422:203-225 (1990); and plays an important role in CNS plasticity and learning. Collingridge, et al., J. Physiol. (Lond). 334:33-46 (1983); Collingridge and Singer, Trends. Pharmacol. Sci. 11:290-296 (1990); and Gustafsson, et al., J. Neurosci. 7:774-780 (1987). Recently, three classes of neuronal glutamate receptors have been cloned, sequenced, and reconstituted in expression vectors. Moriyoshi, et al., Nature 354:31-37 (1991); Monyer, et al., Science 256: 1217-1221 (1992); Sugihara, et al., Biochem. Biophys. Res. Corn. 185:826-832 (1992); Megufo, et al., Nature 357:70-74 (1992); Boulter, et al., Science 249: 1033-1037 (1990); Keinanen, et al., Science 249: 556-560 (1990); and Sommer, et al., Science 249:1580-1585 (1990). One class, metabotropic glutamate receptors, transduce signals to the cell interior by a G-protein coupled mechanism. The other two classes, N-methyl-D-aspartate (NMDA) receptors and non-NMDA, or .alpha.-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate, receptors, are transplasma membrane channels permeable to Na.sup.+, K.sup.+, and Ca.sup.++. Entry of excessive Ca.sup.++ into the cell via intense activation of NMDA receptors, as occurs during brain ischemia, activates mechanisms which cause neuronal death. Rothman, J. Neurosci. 4:1884-1891 (1984); Olney, Adv. Exp. Med. Biol. 203:631-645 (1986); Choi, et al., J. Neurosci. 7:357-368 (1987); and Choi, Neuron. 1:623-634 (1988).
Prior studies of the physiological properties of NMDA receptors, and of glutamate excitotoxicity, depended largely upon the use of primary cultures of neurons prepared from immature rodent. Rothman, J. Neurosci. 4:1884-1891 (1984); Olney, Adv. Exp. Med. Biol. 203:631-645 (1986); Choi, et al., J. Neurosci. 7:357-368 (1987); Choi, Neuron. 1:623-634 (1988); Nowak, et al., Nature 307:462-465 (1984); Mayer, et al., Nature 309:261-263 (1984); Johnson & Ascher, Nature 325:529-531 (1987); and Patneau & Mayer, J. Neurosci. 10:2385-2399 (1990). Many neuron-like cell lines have been screened for glutamate receptor channels but glutamate receptor channels have not been reported. For example, glutamate excitotoxicity has been demonstrated in the neuroblastoma-embryonic retinal hybrid cell line, N18-RE-105, but this is not mediated via NMDA receptor-channels. Murphy, et al., Br. Res. 444:325-332 (1988); Murphy, et al., Br. Res. 460:155-160 (1988); and Murphy, et al., Neuron. 2:1547-1558 (1989). The HCN-1A cell line, established from human cerebral cortical neurons, stains positively for glutamate, but glutamate receptor channels have not been reported in HCN-1A cells. Ronnett, et al., Science 248, 603-605 (1990). Other neuronal cell lines, such as HT-4, demonstrate NMDA-sensitive neurotransmitter release; Morimoto & Koshland, Proc. Natl. Acad. Sci. USA 87:3518-3521 (1990); and NCB-20, a mouse neuroblastoma-Chinese hamster brain hybrid cell line, contain mRNA encoding the NMDA/phencyclidine receptor. Lerma, et al., Proc. Natl. Acad. Sci. USA 86:1708-1711 (1989). However, direct electrophysiological recordings, which are the signature of functional glutamate receptor channels, have not demonstrated NMDA receptor-channels in any neuronal cell line.
A cell line of human neurons suitable for pharmacological and molecular biological studies of neuronal glutamate receptor channels has not been available. Many neuronal cell lines have been screened for glutamate receptor channels, but direct electrophysiological recordings have not demonstrated NMDA receptor channels in any of these lines.